Cytotoxicity of three graphene-related materials in rainbow trout primary hepatocytes is not associated to cellular internalization.

As a consequence of increasing production and use of graphene-related materials (GRM), their release into the aquatic environment is likely to be expected. Development of appropriate model systems to assess their potential toxicity toward aquatic organisms is undoubtedly needed. Of particular relevance are primary cultures of fish hepatocytes, since they maintain similar functionalities as those of the original tissue. Isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) were exposed to ranges of concentrations of different forms of GRM, two graphene oxides (GO) of sheet-like structure and one tubular-shaped carbon nanofiber (CNF) in the presence or absence of fetal bovine serum (FBS) for 24 and 72 h. Metabolic activity, cell membrane integrity, lysosomal function, reactive oxygen species (ROS) formation and interaction with cytochrome P450 1 A enzyme were assessed by using AlamarBlue, 5-carboxyfluorescein diacetate-acetoxymethyl ester, neutral red uptake, dichlorofluorescein and 7-ethoxyresorufin-O-deethylase (EROD) assays, respectively. In the presence of FBS, GO affected metabolic activity and cell membrane integrity more than CNF, whilst absence of serum further reduced cell viability in GRM-exposed cells. GRM did not alter lysosomal function nor did it induce ROS formation or EROD activity. Intracellular uptake was observed only in the case of CNF when incubated without FBS. Primary hepatocytes from rainbow trout appear to be a suitable model to screen for cytotoxicity and to reveal any interaction with GRM. Results emphasize the role of serum proteins in the toxicological responses following exposure to GRM with important implications for the environmental risk assessment of these nanomaterials.

Environmental Impacts by Fragments Released from Nanoenabled Products: A Multiassay, Multimaterial Exploration by the SUN Approach.

Nanoenabled products (NEPs) have numerous outdoor uses in construction, transportation or consumer scenarios, and there is evidence that their fragments are released in the environment at low rates. We hypothesized that the lower surface availability of NEPs fragment reduced their environmental effects with respect to pristine nanomaterials. This hypothesis was explored by testing fragments generated by intentional micronisation ("the SUN approach"; Nowack et al. Meeting the Needs for Released Nanomaterials Required for Further Testing: The SUN Approach. Environmental Science & Technology, 2016 (50), 2747). The NEPs were composed of four matrices (epoxy, polyolefin, polyoxymethylene, and cement) with up to 5% content of three nanomaterials (carbon nanotubes, iron oxide, and organic pigment). Regardless of the type of nanomaterial or matrix used, it was observed that nanomaterials were only partially exposed at the NEP fragment surface, indicating that mostly the intrinsic and extrinsic properties of the matrix drove the NEP fragment toxicity. Ecotoxicity in multiple assays was done covering relevant media from terrestrial to aquatic, including sewage treatment plant (biological activity), soil worms (Enchytraeus crypticus), and fish (zebrafish embryo and larvae and trout cell lines). We designed the studies to explore the possible modulation of ecotoxicity by nanomaterial additives in plastics/polymer/cement, finding none. The results support NEPs grouping by the matrix material regarding ecotoxicological effect during the use phase. Furthermore, control results on nanomaterial-free polymer fragments representing microplastic had no significant adverse effects up to the highest concentration tested.

Negligible cytotoxicity induced by different titanium dioxide nanoparticles in fish cell lines.

Titanium dioxide nanoparticles (TiO2-NPs) have a wide number of applications in cosmetic, solar and paint industries due to their photocatalyst and ultraviolet blocking properties. The continuous increase in the production of TiO2-NPs enhances the risk for this manufactured nanomaterial to enter water bodies through treated effluents or agricultural amendments. TiO2-NPs have shown very low toxicity in a number of aquatic organisms. However, there are no conclusive data about their deleterious effects and on their possible mechanisms of toxic action. At this level, in vitro cell culture systems are a useful tool to gain insight about processes underlying the toxicity of a wide variety of substances, including nanomaterials. Differences in the physiology of different taxa make advisable the use of cells coming from the taxon of interest, but collecting data from a variety of cellular types allows a better understanding of the studied processes. Taking all this into account, the aim of the present study was to assess the toxicity of three types of TiO2-NP, rutile hydrophobic (NM-103), rutile hydrophilic (NM-104) and rutile-anatase (NM-105), obtained from the EU Joint Research Centre (JRC) repository, using various fish cell lines (RTG-2, PLHC-1, RTH-149, RTL-W1) and rainbow trout primary hepatocytes. For comparative purposes, the effect of different dispersion protocols, end-point assays and extended exposure time was studied in a fish cell line (RTG-2) and in the rat hepatoma cell line (H4IIE). TiO2-NPs dispersions showed a variable degree of aggregation in cell culture media. Disruption of mitochondrial metabolic activity, plasma membrane integrity and lysosome function was not detected in any cell line after exposure to TiO2-NPs at any time and concentration ranges tested. These results are indicative of a low toxicity of the TiO2-NPs tested and show the usefulness of fish cells maintained in vitro as high throughput screening methods that can facilitate further testing in the framework of integrated testing strategies.

Induction of EROD and BFCOD activities in tissues of barbel (Barbus callensis) from a water reservoir in Algeria.

EROD and BFCOD activities were measured in liver and gills of barbel (Barbus callensis, a native North African species) captured at Beni Haroun lake, the most important water reservoir in Algeria. This lake receives wastewater from different origins. Thus, we assessed the level of pollution through the induction of detoxification activities in tissues of barbel, evaluating simultaneously the suitability of this species to be used as a sentinel. Fish were collected between March 2015 and January 2016 at three locations taking into account the pollution sources and accessibility. In liver, EROD and BFCOD showed the highest induction in October specially in the location of the dam that received pollutants. In gills, only EROD, but not BFCOD, activity was detected. Maximal EROD induction was noted in samples from January. Fish cell lines (RTG-2 and PLHC-1) were exposed to sediments extracts collected at Beni Haroun lake and enzyme activities (EROD and BFCOD, respectively) were measured. Sediment extracts did not induce BFCOD activity. The EROD induction observed in RTG-2 cells was in line with the results observed in fish tissues. Our results suggest that the lake is at risk from pollution and that Barbus callensis is a good sentinel species.

Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.

Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (ß-naphthoflavone (ß-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on ß-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard.

Detection of effects caused by very low levels of contaminants in riverine sediments through a combination of chemical analysis, in vitro bioassays, and farmed fish as sentinel.

Aquatic organisms are often exposed to mixtures of low levels of pollutants whose presence and effects can pass easily unnoticed if only traditional monitoring strategies are employed. The main aim of this work was to assess the presence and effects of trace levels of pollutants in a scarcely affected area through the combination of chemical and biological approaches. Sediments were collected along a river with little anthropogenic pressure and assayed for cytochrome P450 (Cyp1a)-dependent ethoxyresorufin-O-deethylase (EROD) activity with the rainbow trout gonadal cell line RTG-2. Chemical analyses were performed in these sediments using two-dimensional gas chromatography-time-of-flight mass spectrometry. Sediment samples induced EROD activity, and chemical analyses evidenced the presence of a wide variety of contaminants in the range of nanograms per gram of dry weight. Correlation analysis between EROD induction and chemical analyses data showed an r value of 0.840 (p < 0.05). In addition, fish from a fish farm located downstream of the sampling points exhibited high hepatic EROD levels as well as an induced expression of cyp1a and cyp3a. In conclusion, only an appropriate combination of biological and chemical techniques allowed the detection of the presence of trace levels of contaminants in a theoretically nonaffected river.

Chlorotriazines do not activate the aryl hydrocarbon receptor, the oestrogen receptor or the thyroid receptor in in vitro assays.

Atrazine, prometryn, propazine and simazine are chlorotriazines that are commonly employed as herbicides. However, their use is a major cause of concern, due to their reported endocrine disrupting effects in different taxa. Data from studies on the molecular and cellular processes underlying the hormonal action of these substances are contradictory. The ability of these chlorotriazines and the atrazine metabolites, desethyl-s-chlorotriazine and desisopropyl-s-chlorotriazine, to trigger responses mediated by the oestrogen receptor (ER), aryl hydrocarbon receptor (AhR) and thyroid receptor (TR), was studied by using in vitro approaches. Transcriptional activation assays were applied to observe the activation of ER and TR. The induction of ethoxyresorufin-O-deethylase (EROD) activity in the RTG-2 cell line served as an indicator of AhR activation. No responses were found in any of the assays, with any of the six chlorotriazines tested. Our observations indicate that the chlorotriazines tested are unlikely to cause their endocrine effects via these receptors.

Testing the Aquatic Toxicity of 2D Few-Layer Graphene Inks Using Rainbow Trout (Oncorhynchus mykiss): In Vivo and In Vitro Approaches to Support an SSbD Assessment.

Graphene-based conductive inks offer attractive possibilities in many printing technology applications. Often, these inks contain a mixture of compounds, such as solvents and stabilizers. For the safe(r) and sustainable use of such materials in products, potentially hazardous components must be identified and considered in the design stage. In this study, the hazards of few-layer graphene (FLG)-based ink formulations were tested in fish using in vitro (RTL-W1 cell line) and in vivo aquatic ecotoxicity tests (OECD TG 203). Five ink formulations were produced using different processing steps, containing varying amounts of solvents and stabilizers, with the end products formulated either in aqueous solutions or in powder form. The FLG ink formulations with the highest contents of the stabilizer sodium deoxycholate showed greater in vitro cytotoxic effects, but they did not provoke mortality in juvenile rainbow trout. However, exposure led to increased activities of the cytochrome P450 1a (Cyp1a) and Cyp3a enzymes in the liver, which play an essential role in the detoxification of xenobiotics, suggesting that any effects will be enhanced by the presence of the stabilizers. These results highlight the importance of an SSbD approach together with the use of appropriate testing tools and strategies. By incorporating additional processing steps to remove identified cytotoxic residual solvents and stabilizers, the hazard profile of the FLG inks improved, demonstrating that, by following the principles of the European Commission's safe(r) and sustainable by design (SSbD) framework, one can contribute to the safe(r) and sustainable use of functional and advanced 2D materials in products.

Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2.

BACKGROUND: Graphene and graphene derivative nanoplatelets represent a new generation of nanomaterials with unique physico-chemical properties and high potential for use in composite materials and biomedical devices. To date little is known about the impact graphene nanomaterials may have on human health in the case of accidental or intentional exposure. The objective of this study was to assess the cytotoxic potential of graphene nanoplatelets with different surface chemistry towards a human hepatoma cell line, Hep G2, and identify the underlying toxicity targets. METHODS: Graphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelet suspensions were obtained in water and culture medium. Size frequency distribution of the suspensions was determined by means of dynamic light scattering. Height, lateral dimension and shape of the nanoplatelets were determined using atomic force and electron microscopy. Cytotoxicity of GO and CXYG nanoplatelets was assessed in Hep G2 cells using a battery of assays covering different modes of action including alterations of metabolic activity, plasma membrane integrity and lysosomal function. Induction of oxidative stress was assessed by measuring intracellular reactive oxygen species levels. Interaction with the plasma membrane, internalization and intracellular fate of GO and CXYG nanoplatelets was studied by scanning and transmission electron microscopy. RESULTS: Supplementing culture medium with serum was essential to obtain stable GO and CXYG suspensions. Both graphene derivatives had high affinity for the plasma membrane and caused structural damage of the latter at concentrations as low as 4 µg/ml. The nanoplatelets penetrated through the membrane into the cytosol, where they were concentrated and enclosed in vesicles. GO and CXYG accumulation in the cytosol was accompanied by an increase in intracellular reactive oxygen species (ROS) levels, alterations in cellular ultrastructure and changes in metabolic activity. CONCLUSIONS: GO and CXYG nanoplatelets caused dose- and time-dependent cytotoxicity in Hep G2 cells with plasma membrane damage and induction of oxidative stress being important modes of toxicity. Both graphene derivatives were internalized by Hep G2, a non-phagocytotic cell line. Moreover, they exerted no toxicity when applied at very low concentrations (< 4 µg/ml). GO and CXYG nanoplatelets may therefore represent an attractive material for biomedical applications.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

The Potentiating Effect of Graphene Oxide on the Arylhydrocarbon Receptor (AhR)-Cytochrome P4501A (Cyp1A) System Activated by Benzo(k)fluoranthene (BkF) in Rainbow Trout Cell Line.

The increasing use of graphene oxide (GO) will result in its release into the environment; therefore, it is essential to determine its final fate and possible metabolism by organisms. The objective of this study was to assess the possible role of the aryl hydrocarbon receptor (AhR)-dependent cytochrome P4501A (Cyp1A) detoxification activities on the catabolism of GO. Our hypothesis is that GO cannot initially interact with the AhR, but that after an initial degradation caused by other mechanisms, small fractions of GO could activate the AhR, inducing Cyp1A. The environmental pollutant benzo(k)fluoranthene (BkF) was used for the initial activation of the AhR in the rainbow trout (Oncorhynchus mykiss) cell line RTL-W1. Pre-, co-, and post-exposure experiments with GO were performed and Cyp1A induction was monitored. The strong stimulation of Cyp1A observed in cells after exposure to GO, when BkF levels were not detected in the system, suggests a direct action of GO. The role of the AhR was confirmed by a blockage of the observed effects in co-treatment experiments with αNF (an AhR antagonist). These results suggest a possible role for the AhR and Cyp1A system in the cellular metabolism of GO and that GO could modulate the toxicity of environmental pollutants.

VKORC1 single nucleotide polymorphisms in rodents in Spain.

Rodents are considered one of the animal pests with the greatest impact on agricultural production and public health. Anticoagulant rodenticides (ARs), used as one of the most effective ways to control rodent populations worldwide, inhibit the vitamin K 2,3-epoxide reductase (VKORC1) enzyme involved in blood coagulation. Resistances to ARs are mainly associated with mutations or single nucleotide polymorphisms (SNPs) in the vkorc1 gene. Since the information on this subject is scarce in Spain, we monitored and discovered rodent SNPs that could favour genetic resistance in its populations. For that, more than 200 samples of stools and tails from brown rat (Rattus norvegicus), black rat (Rattus rattus) and mouse (Mus musculus) were collected from 12 Spanish regions previously identified with low AR efficacy in coordination with the National Association of Environmental Sanitation Companies (ANECPLA) and the managing entities of four locations. We then sequenced their vkorc1 exon 3 corresponding genomic DNA. We identified genotypic vkorc1 variations corresponding to amino acid changes at the VKORC1 protein at the S149I - S149T and the E155K - E155Q mutations, depending on the rodent species. Computational analysis of binding predictions found out that the brown rat S149I mutation predicted a high reduction of the binding affinity of chlorophacinone and brodifacoum ARs while, the black rat S149T, E155K and E155Q mutations slightly reduced bromadiolone AR binding. These results suggest that these mutations may be one of the causes of the increased resistance to those ARs.

Liver biomarkers response of the neotropical fish Aequidens metae to environmental stressors associated with the oil industry.

The Acacias River in Colombia receives large volumes of industrial effluents mostly derived from the oil industry. To contribute to the study of the possible effects of industrial wastewaters on the aquatic environment and particularly on fish populations, a native neotropical fish, Aequidens metae was used as a sentinel species. Wild specimens of A. metae were caught at three different places of the Acacias River taking as reference the point of discharge of an oil industry effluent; upstream, downstream, and at the vicinity of the discharge pipe. A fourth sampling site was chosen as a reference site away from urban settlements. Samplings were performed twice, during the rainy and dry seasons. After anesthesia animals were weighted and measured, and humanely sacrificed. Livers were extracted, frozen on site and transported to the laboratory. Condition indices were calculated. Total protein content and the detoxification 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity were estimated. Histopathological alterations were also evaluated. Water quality was estimated through the measurement of several variables. Results obtained evidenced that the highest induction in EROD activity and the strongest histological alterations in liver of the monitored fish appeared during the dry seasons at the discharge site and downstream to this point.

Populus alba L., an Autochthonous Species of Spain: A Source for Cellulose Nanofibers by Chemical Pretreatment.

In order to identify new sustainable sources for producing cellulose nanofibers (CNFs), fast-growing poplar (Populus alba L.) wood was evaluated herein. For that purpose, bleached poplar kraft pulp was produced and submitted to TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) mediated oxidation (TEMPO-ox) chemical pretreatment followed by microfluidization. The resulting CNFs were thoroughly characterized, including a rheological study at different pH values. Poplar CNFs showed properties comparable to eucalypt CNFs (reference material for CNFs production), showing high carboxylate content (1048 ± 128 µmol g-1), fibrillation yield (87.3% ± 8.1%), optical transmittance (83% at 700 nm) and thermal stability (up to more than 200 °C). Regarding the rheological study, whereas pH from 4 to 10 did not produce significant changes in rheological behavior, a reduction of pH down to 1 led to an order-of-magnitude increase on the viscoelastic functions. Therefore, poplar CNF shows potential in the pH-sensitive hydrogels application field. Finally, the possible ecotoxicity of poplar CNF was assessed. The decrease in cell viability was very low so that only concentrations causing a 10% cytotoxicity could be calculated for the assay detecting alterations in cell metabolism (10 µg mL-1) and plasma membrane integrity (60 µg mL-1).

Usefulness of fish cell lines for the initial characterization of toxicity and cellular fate of graphene-related materials (carbon nanofibers and graphene oxide).

Graphene-related materials (GRMs) are one of the most attractive materials from an application perspective, consequently their release into aquatic environments is highly likely. In the present work, the potential of fish hepatocytes (topminnow fish hepatoma cell line, PLHC-1) and macrophages (carp leukocyte cell line, CLC) to study the toxicity and intracellular fate of helical-ribbon carbon nanofibers (CNFs) and graphene oxide (GO) used in a variety of intermediate industrial products was evaluated, allowing a first ranking of GRMs according to their cytotoxicity. Cells were exposed to a concentration range of 0-200 µg ml-1 of GRMs for 24 and 72 h and cell viability was assessed by measuring mitochondrial activity (AlamarBlue assay), plasma membrane integrity (5-carboxyfluorescein diacetate-acetoxymethyl ester assay) and lysosomal function (neutral red uptake assay). Results showed that both the cell type and the choice of endpoint determined the toxicity of GRMs. In both cell lines, CNFs appeared to have higher toxicity than GO and the highest degree of graphitization in fibers was associated with lower toxicity. Transmission electron microscopy revealed that CNFs were taken up into membrane-bound compartments of PLHC-1 cells in a size-independent manner, whereas in CLC, longer CNFs were encountered free in the cytoplasm and only the shorter CNFs were localized in membrane-surrounded vesicles. GO sheets were present within vesicles as well as free in the cytoplasm of both cell types. These findings contribute to the understanding of the toxicity and behaviour of these GRMs in living systems, therefore aiding in designing safer materials for the environment.

Investigating the Impact of Manufacturing Processes on the Ecotoxicity of Carbon Nanofibers: A Multi-Aquatic Species Comparison.

Manufactured nanomaterial production is outpacing the ability to investigate environmental hazard using current regulatory paradigms, causing a backlog of materials requiring testing. To ameliorate this issue, regulatory bodies have proposed integrating safety into the production of novel nanomaterials, allowing for hazards to be identified early in development rather than aftermarket release. In addition, there is a growing interest in short-term ecotoxicity testing to rapidly identify environmental hazards. In this sense, the present study investigated 3 carbon nanofibers (CNFs), created with different production methods, using short-term in vitro and in vivo exposures on fish cell lines, mussel hemocytes, crustacea, and algae. The present study investigated if differences in ecotoxicity hazard between the CNFs could be identified and, if so, which product could be considered less hazardous. A major challenge in assessing the potential hazards posed by manufactured nanomaterials is standardizing the preparation for testing. Standardized operating protocols have been proposed using protein to facilitate the preparation of stable stock suspension, which is not environmentally representative. As such, the study also assessed the potential impacts these standardized protocols (with or without the use of protein) could have on the interpretation of environmental hazard. The results demonstrated that there were clear differences between the 3 CNFs and that the dispersion protocol influenced the interpretation of hazard, demonstrating a need for caution when interpreting ecotoxicity in a regulatory context. Environ Toxicol Chem 2019;38:2314-2325. © 2019 SETAC.

In-vitro screening of the antiestrogenic activity of chemicals.

BACKGROUND: Many chemicals have the potential to interfere with the endocrine systems of humans and wildlife, leading to adverse health effects. In the tiered testing strategies developed for regulatory hazard assessment, in-vitro screens could serve for prioritisation of compounds and for guiding subsequent testing. OBJECTIVE: To describe in-vitro assays to detect antiestrogenic activity of chemicals. METHODS: Antiestrogenicity was considered in this review as any inhibition or reduction of estrogen-induced processes due to interference with the normal functioning of the estrogen receptor pathway. Accordingly, in-vitro screening assays for antiestrogenicity have to consider all the possible mechanisms by which this inhibition may occur. Such assays include binding assays, cell proliferation assays, reporter gene assays, and gene activation/protein production assays. RESULTS/CONCLUSIONS: While binding assays appear to be of limited value in assessing antiestrogenicity, assays using differentiated cells with metabolic competence and a varied receptor/regulatory factor equipment have the capability to detect various modes of antiestrogenic action.

Modulation of aryl hydrocarbon receptor transactivation by carbaryl, a nonconventional ligand.

Carbaryl (1-naphthyl-N-methylcarbamate), a widely used carbamate insecticide, induces cytochrome P450 1A gene expression in mammalian cells. This activity is usually mediated by the interaction of the compound with the aryl hydrocarbon receptor. However, it has been proposed that this mechanism does not apply to carbaryl because its structure differs from that of typical aryl hydrocarbon receptor ligands. We show here that carbaryl promotes activation of target genes in a yeast-based bioassay expressing both aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator. By contrast, carbaryl acted as a competitive inhibitor, rather than as an agonist, in a simplified yeast system, in which aryl hydrocarbon receptor nuclear translocator function is bypassed by fusing aryl hydrocarbon receptor to a heterologous DNA binding domain. This dual action of carbaryl, agonist and partial antagonist, was also observed by comparing carbaryl response in two vertebrate cell lines. A yeast two-hybrid assay showed that the mammalian coactivator cAMP response element-binding protein readily interacts with aryl hydrocarbon receptor bound to its canonical ligand beta-naphthoflavone, but not with the carbaryl-aryl hydrocarbon receptor complex. We propose that carbaryl interacts with aryl hydrocarbon receptor, but that its peculiar structure imposes a substandard configuration on the aryl hydrocarbon receptor ligand-binding domain that prevents interaction with key coactivators and activates transcription without the need for aryl hydrocarbon receptor nuclear translocator. This effect may be relevant in explaining its physiological effects in exposed animals, and may help to predict its effects, and that of similar compounds, in humans. Our data also identify the aryl hydrocarbon receptor/cAMP response element-binding protein interaction as a molecular target for the identification and development of new aryl hydrocarbon receptor antagonists.

Remediation efficiency of three treatments on water polluted with endocrine disruptors: Assessment by means of in vitro techniques.

Chemical substances with potential to disrupt endocrine systems have been detected in aquatic environments worldwide, making necessary the investigation about water treatments able to inhibit such potential. The present work aimed to assess the efficiency for removing endocrine disruptors (with estrogenic and androgenic activity) of three simple and inexpensive substrates that could be potentially used in sectors or regions with limited resources: powdered activated carbon (PAC), powdered natural zeolite (ZEO) (both at a concentration of 500 mg L-1) and natural aquatic humic substances (AHS) (at 30 mg L-1). MilliQ-water and mature water from fish facilities (aquarium water, AW), were artificially spiked with 17ß-estradiol (E2), 17α-ethinylestradiol and dihydrotestosterone. Moreover, effluent samples from waste water treatment plants (WWTP) were also submitted to the remediation treatments. Estrogenic and androgenic activities were assessed with two cell lines permanently transfected with luciferase as reporter gene under the control of hormone receptors: AR-EcoScreen containing the human androgen receptor and HER-LUC transfected with the sea bass estrogen receptor. PAC was efficiently removing the estrogenic and androgenic compounds added to milliQ and AW. However, androgenic activity detected in WWTP effluents was only reduced after treatment with ZEO. The higher surface area of PAC could have facilitated the removal of spiked hormones in clean waters. However, it is possible that the substances responsible of the hormonal activity in WWTP have adsorbed to micro and nanoparticles present in suspension that would have been retained with higher efficiency by ZEO that show pores of several microns in size.